Radioactive transition metal-imido hetero-diphosphine complexes, their preparation and radiopharmaceutical compositions thereof

ABSTRACT

The present invention provides radioactive metal heterocomplexes of formula (I): [(Me═N—R)L 1 L 2 ]+Z. (I), wherein Me, R, L 1  L 2  and Z −  have the meanings indicated in the description. The complexes include a trivalent radioactive metal-imido group, typically a technetium- or rhenium-imido group, strongly stabilized by the presence of an ancillary tridentate hetero-diphosphine ligand L 1 , which allows the formation of substitution-inert [(Me═N—R)L 1 ] moieties. Such moieties are fixed in an intermediate [(Me═N—R)Y 2 L 1 )] +  compound which contains two labile, cispositioned, Y ligands, where Y is preferably an halide group. The latter are easily replaced by a bidentate ligand L 2  to give the final [(Me═N—R)L 1 L 2 ] + Z − heterocomplexes. The complexes of the invention are useful for the preparation of radiopharmaceuticals: in fact, a bioactive fragment which confers biological target-seeking properties can be introduced either on the L 2  framework or the imido R group.

FIELD OF THE INVENTION

The present invention relates to a radioactive transition metal-imidoheterocomplex, a radiopharmaceutical comprising said complex and aprocess for producing thereof. More particularly, the complex of theinvention comprises an imido derivative of radioactive technetium orrhenium and two different ligands coordinated therewith.

STATE OF THE ART

The ^(99m)Tc-imido core has been only generically cited among a seriesof new cores suitable for the development of technetiumradiopharmaceuticals (Archer C. et al. WO 91/03262 and referencestherein). Despite a few examples obtained at “carrier added (ca)” or, inother words, macroscopic level, using ⁹⁹Tc and cold Re, in which thepresence of the imido group was clearly established, the transfer ofthis chemistry at “no carrier added (nca)” or, in other words,microscopic level, has met severe obstacles so far. The terms“macroscopic” or “carrier added” level refer to chemical reactionsoccurring at concentrations ranging from 10⁻³ to 10⁻⁵ M. Reactions areperformed in conventional laboratories (if necessary approved for lowlevel radioactivity) using from 1 to 200 mg amounts of non-radioactiveRe or radioactive ⁹⁹Tc. The terms “microscopic” or “no carrier added”level refer to reactions occurring at concentrations ranging from 10⁻⁶to 10⁻⁹ M with micrograms to nanograms amounts of radioactive ^(99m)Tcor of radioactive ¹⁸⁸Re (see also IUPAC Compendium of ChemicalTerminology, 2nd Edition (1997), wherein the following definition isprovided for “no carrier added”: “ . . . a preparation of a radioactiveisotope which is essentially free from stable isotopes of the element inquestion . . . ”). These reactions are routinely performed in hospitalNuclear Medicine Departments for clinical purposes (Deutsch, E., Libson,K., Recent advances in technetium chemistry: bridging inorganicchemistry and nuclear medicine, Comments Inorg. Chem., 3, 83-103, 1984).

Working at macroscopic level with ⁹⁹Tc, only five crystal structures ofTc(V)-imido species have been reported. They include [Tc(NR)Cl₃(PPh₃)₂],where R is phenyl (Nicholson T. et al. Inorg. Chim. Acta, 187 (1991) 51)or tolyl (Dilworth J. et al. in Technetium in Chemistry and NuclearMedicine—3, Raven Press, New York, (1990), 109). By varying thebulkiness of both the halide (from Cl to Br) and/or the phosphine (fromPPh₃ to PMePh₂ and PMe₂Ph), three additional phenyl-imido compounds havebeen produced, i.e.: [Tc(NPh)Cl₃(PMePh₂)₂], [Tc(NPh)Br₃(PMePh₂)₂](Rochon F. D. et al. Inorg. Chem. 34 (1995) 2273) and[Tc(NPh)Cl₂(PMe₂Ph)₃]⁺ (Nicholson T. et al. Inorg. Chim. Acta, 230(1995) 205).

Nevertheless, no specific disclosure, nor even suggestions, have everbeen made on the possibility of successfully transferring the abovepreparations also at microscopic level.

All the above species are six-coordinated compounds with slightdistortion from the ideal octahedron. The co-ordination sphere istotally filled with monodentate ligands (halides and monophosphines)that do not impose heavy steric constraints. In this connection the Tcatom moves off the mean basal plane towards the imido unit by only 0.11Å. The imido core is essentially linear (mean Tc═N—C angle 175.70) witha marked portion of the Tc—N bond showing double bond character (meanvalue 1.71 Å; see G. Bandoli et al., Coord. Chem. Rev., 214 (2001) 43).

The possibility to introduce chelate ligands in the co-ordination sphereof Tc(V)-imido compounds was demonstrated separately by differentauthors. Nicholson et al. (Inorg. Chim. Acta, 196 (1992) 27) introduceda simple bidentate diphosphine, i.e. 1,2-bis (diphenylphosphine)ethane(dppe) to yield [Tc(NPh)Cl₃(dppe)]. Tisato et al. (J. Organom. Chem.637-639 (2001) 772) utilised the 1,2-bis (diphenylphosphine)ferrocenylresidue (dppf) to produce the analogous [Tc(NPh)Cl₃(dppf)] complex.Similarly, rhenium (Re) complexes where synthesized, by using thebidentate (2-diphenylphosphine)benzeneamine (H₂dpa), or the tetradentateN,N′-bis[(2-diphenylphosphine)phenyl]propane-1,3-diamine (Refosco F. etal, J. Chem. Soc. Dalton Trans 1998, 923-930).

Notwithstanding the above results, successive extensive efforts tostabilize the ^(99m)Tc-imido core have surprisingly failed, because noappropriate combinations of donor atoms was found to support this group,while, on the contrary, this target was successfully achieved in thecase of the somewhat similar ^(99m)Tc-oxo and ^(99m)Tc-nitrido cores.Oxo [^(99m)Tc(O)]³⁺, imido [^(99m)Tc(NR)]³⁺ and nitrido [^(99m)Tc(N)]²⁺moieties are isoelectronic cores, the metal being in the 5⁺ oxidationstate, and, in this connection, the technetium-imido group can beconsidered as intermediate between oxo and nitrido cores. Previousinvestigations have established that Tc(V)-oxo groups are readilystabilized by tetradentate ligands, as shown by N₄-hexamethylpropyleneamine oxime (HM-PAO), N₃S-mercaptoacetyltriglycyl (MAG₃) andN₂S₂-ethylcysteine dimer (ECD) in the clinically used Ceretec®,Technescan® and Neurolite® radiopharmaceuticals. On the other hand, theTc(V)-nitrido group prefers a combination of two different polydentateligands. Previous studies on ⁹⁹Tc-imido complexes have shown thatdistorted octahedral environments are usually achieved, the coordinationbeing supported by monodentate ligands, preferably tertiarymonophosphines and halides. Attempts to replace the monodentate P-baseddonors present in the prototype precursor [Tc(NPh)Cl₃(PPh₃)₂] withvarious polydentate chelates were not successful, thus indicating thatthe imido group should be stabilized only by the presence of monodentatemonophosphine ligands.

DESCRIPTION OF THE INVENTION

It has now surprisingly been found that it is possible to replace thetwo monodentate triphenylphosphine substituents with a tridentateheterodiphosphine ligand L¹ which comprises an electron donor heteroatomin the spacer chain linking said two phosphine groups, without affectingthe stability of the imido group in the resulting [(Me═N—R)Y₂(L¹)]⁺Y⁻compounds, wherein Y is a leaving group, such as a halogen atom, anhydroxy or an alkoxy group. The facial configuration of the tridentatehetero-diphosphine chelate induces a cis-coordination of the two Ygroups, which in turn can be easily substituted with suitable bidentateligands L² to give the final desired [(Me═N—R)L¹L²]⁺Z⁻ metalheterocomplex.

Accordingly, the present invention primarily provides a radioactivetransition metal-imido hetero-diphosphine complex compound of formula(I):[(Me═N—R)L¹L²]⁺Z⁻  (I),wherein:Me is a radioactive transition metal selected from the group consistingof ^(99m)Tc, ¹⁸⁶Re, ¹⁸⁸Re;R is a C₁-C₁₅ linear or branched alkyl or alkenyl residue, optionallyinterrupted by —O—, —S—, —N(R′)—, where R′ is H or C₁-C₆ alkyl, and/oroptionally substituted with halogen, hydroxy, C₁-C₅ alkoxy, carboxy,ester, thiol, primary or secondary amino or amido groups, or R is phenylor an aryl residue, being R optionally substituted with a biologicallyactive substance, wherein said biologically active substance is selectedamong sugars, amino acids, fatty acids, vitamins, hormones, peptides,catecholamines, said catecholamines being optionally conjugated, viapeptidic bond, to the other above mentioned biologically activesubstances;

L¹ is a tridentate hetero-diphosphine ligand of formula (II):

wherein:R¹, R², R³ and R⁴, which may be the same or different, have the samemeanings as R;X is oxygen, sulphur, NR⁵, wherein R⁵ is hydrogen or R;n is an integer ranging from 1 to 5;L² is a bidentate ligand, which comprises a combination of two donoratoms, selected from the group consisting of oxygen, sulphur andnitrogen, said atoms being preferably negatively charged and beingseparated by a spacer of 2 to 4 members, said spacer being an aliphaticchain or part of an aromatic ring, L² being optionally conjugated to abiologically active substance as above defined;Z⁻ is a mononegative counter-ion selected from the group consisting ofCl⁻, Br⁻, OH⁻, ClO₄ ⁻, alkoxylate, preferably EtO⁻, tetrafluoroborate.

Preferred R are methyl, ethyl, propyl, isopropyl, butyl, isobutyl,octyl, decyl, dodecyl, propenyl, butenyl, pentenyl, phenyl, benzyl,tolyl, 4-methoxy-benzyl, 4-ethoxy-benzyl, salicyl.

Preferred tridentate hetero-diphosphine ligands L¹ of formula (II) arethose where n=2. Most preferred ligands L¹ are selected from the groupconsisting of:

(C₆H₅)₂PCH₂CH₂N(H)CH₂CH₂P(C₆H₅)₂;

(C₆H₅)₂PCH₂CH₂N(CH₃)CH₂CH₂P(C₆H₅)₂;

(CH₃)₂PCH₂CH₂N(CH₃)CH₂CH₂P(CH₃)₂;

(C₆H₅)₂PCH₂CH₂SCH₂CH₂P(C₆H₅)₂;

(C₆H₅)₂PCH₂CH₂OCH₂CH₂P(C₆H₅)₂;

(C₆H₅)₂PCH₂CH₂N(CH₂CH₂OCH₃)CH₂CH₂P(C₆H₅)₂.

Bidentate ligands L² preferably comprise a 2 or 3 membered spacer asdefined above between the two electron-donor atoms. Suitablecombinations of electron-donor atoms, preferably negatively charged, are[O⁻,O⁻], [N⁻,O⁻], [S⁻,O⁻], [N⁻,N⁻], [N⁻,S⁻] and [S⁻,S⁻]. Preferredbidentate ligands are catecholate⁽²⁻⁾; carbonate⁽²⁻⁾;1,2-aminophenolate⁽²⁻⁾; 1,2-benzenedithiolate⁽²⁻⁾;ethyleneglycolate⁽²⁻⁾; ethylenediaminate⁽²⁻⁾; ethylenedithiolate⁽²⁻⁾;1,2-phenylenediaminate⁽²⁻⁾; 1,2-aminothiophenolate⁽²⁻⁾;thiosalicylate⁽²⁻⁾; 1,2-aminoethanolate⁽²⁻⁾ and the like.

The bidentate ligands L² preferably carry a biologically activesubstance as defined above. Among said biologically active substances,preferred are catecholamines, like dopamine, L-DOPA and3-hydroxythyramine. Catecholamines may, in turn, be conjugated, viapeptidic bond, to other physiologically active substances. In apreferred embodiment of the invention, vitamin H is conjugated todopamine.

The radioactive compounds of formula (I) can be obtained by reacting anintermediate compound of formula (III):[(Me═N—R)Y²(L¹)]⁺Y⁻  (III),with a bidentate ligand L²,wherein Me, R, L¹ and L² are as defined above and Y is halogen,preferably chlorine, or bromine, hydroxy or alkoxy, preferably ethoxy,or a leaving group which easily undergoes nucleophilic substitution.

The reaction is usually carried out in an organic solvent, in thepresence of an organic base, or under buffered conditions, preferably inphosphate buffer. Preferred solvents are alcohols and chlorinatedsolvents. Preferred organic bases are tertiary amines, more preferred istriethylamine. The reaction temperature ranges from room temperature tothe reflux temperature of the solvent.

The final product is separated and purified with conventional techniqueslike salification, crystallization, chromatography as described indetail in the following experimental section.

Intermediate compounds of formula (III) are in turn synthesized asdescribed in Scheme 1 below from oxides of radioactive transitionmetals, preferably ^(99m)TcO₄ ⁻, ¹⁸⁶ReO₄ ⁻, or ¹⁸⁸ReO₄ ⁻, morepreferably ^(99m)TcO₄ ⁻, which are treated with an excess of tertiarymonophosphines, preferably PPh₃, in acidic hydro-alcoholic solutions andin the presence of a suitable imido donor (D), to give an imido complexof formula (IV), wherein Me, R and Y are as defined above. Successivetreatment with an above described ligand L¹ affords the desiredintermediates of formula (III).

Suitable imido donors D according to the invention are preferably1-substituted-2-acetyl hydrazine, most preferably I-phenyl-2-acetylhydrazine (PAH).

The reaction of L¹ with imido complexes of formula (IV) is preferablycarried out in organic solvents like alcohols, chlorinated solvents,acetonitrile or a mixture thereof, optionally in the presence of anorganic base like triethylamine, at a temperature ranging from roomtemperature to the reflux temperature of the solvent. The finalpurification of the desired intermediate of formula (III) is thoroughlydescribed in the following experimental section. It has finally beendemonstrated that the preferred ^(99m)Tc-imido heterocomplexes accordingto the invention can be obtained at microscopical level via a three-stepapproach starting from pertechnetate sodium salt eluted from acommercial ⁹⁹Mo/^(99m)Tc generator. In the first step pertechnetate istreated with an excess of tertiary monophosphine in hydro-alcoholicsolutions acidified with hydrochloric acid and in the presence of1-phenyl-2-acetyl hydrazine. In the second step, addition of thetridentate hetero-diphosphine ligand in an organic solvent affords theintermediate species [^(99m)Tc(NPh)Cl₂(L¹)]⁺Y⁻. By adjusting pH at 7.4with phosphate buffer, and by adding the preferred bidentate ligand L²,the desired imido heterocomplex is produced in high radiochemical yield(see Table 1).

Preferred complexes of the invention are:

[^(99m)Tc(NPh)(PNHP)(O,N-ap)];

[^(99m)Tc(NPh)(PNHP)(O,O-car)];

[^(99m)Tc(NPh)(PNMeP)(O,N-ap)];

[^(99m)Tc(NPh)(PNMeP)(S,N-atp)];

[^(99m)Tc(NPh)(PNMeP)(O,O-cat)];

[^(99m)Tc(NPh)(PNMeP)(S,O-tsal)];

[^(99m)Tc(NPh)(PNMeP)(S,S-bdt)];

wherein

PNHP means (C₆H₅)₂PCH₂CH₂N(H)CH₂CH₂P(C₆H₅)₂;

PNMeP means (C₆H₅)₂PCH₂CH₂N(CH₃)CH₂CH₂P(C₆H₅)₂;

O,N-ap means 2-aminophenolate⁽²⁻⁾;

S,N-atp means 2-aminothiophenolate⁽²⁻⁾;

O,O-cat means catecholate⁽²⁻⁾;

O,O-car means carbonate⁽²⁻⁾;

S,O-tsal means thiosalicylate⁽²⁻⁾;

S,S-bdt means 1,2-benzenedithiolate⁽²⁻⁾

The reactivity of imido-containing species has been thoroughly studiedat macroscopic level using [⁹⁹Tc(NPh)Cl₃(PPh₃)₂] and [Re(NPh)Cl₃(PPh₃)₂]as precursors.

These precursors react with tridentate hetero-diphosphine ligands L¹ toyield intermediate compounds of the type [Me(NPh)Cl₂L¹)][Cl]. Thehetero-diphosphine ligand acts as a tridentate donor due to the presenceof the electron-donor X heteroatom interposed in the diphosphine chainand gives rise to three different octahedral configurations, shown bythe following formulas (IIIA): fac,cis (IIIB) mer,cis and (IIIC)mer,trans.

In formulae (IIIA), (IIIB) and (IIIC) a denotes the positions occupiedby ligand L¹ and b denotes the positions occupied by ligand Y.

The halide group, positioned trans with respect to the imido core in theintermediate mer,cis-[Me(NPh)Cl₂(L¹)]⁺ compounds, easily undergoessubstitution with nucleophilic ligands (such as ethanolate), indicatingthat halide ligands are labile and good leaving groups. In similarsubstitution reactions, cis-positioned halides are replaced by bidentatenucleophilic ligands such as ethyleneglycol or catechol or the like toyield imido heterocomplexes of the type [Me(NPh)L¹L²]⁺Z⁻. Thus, bothmer,cis-[Me(NPh)Cl₂L¹]⁺ and fac,cis-[Me(NPh)Cl₂L¹]⁺ isomers are usefulintermediates for the production of mixed imido heterocomplexes. On thecontrary, the mer,trans-[Me(NPh)Cl₂L¹]⁺ isomers give rise to theheterocomplexes in a negligible yield, as a result of heavy stericconstraints imposed by the meridional coordination of the L¹ diphosphinecombined with the trans-halide configuration. Thus, a reciprocalcis-position of the halide groups in the intermediate compounds is anessential pre-requisite for obtaining heterocomplexes of the invention.

The stereochemistry of the L¹ ligand in the final heterocomplex isalways facial, the bidentate ligand L² filling the residual twopositions on the equatorial plane of the octahedron.

The present invention is illustrated in further detail in the followingexamples.

EXAMPLES

The tridentate hetero-diphosphine ligands L¹ and the bidentate ligandsL² used in the following examples are abbreviated as follows:

Tridentate hetero-diphosphine ligands L¹:

PNHP; (C₆H₅)₂PCH₂CH₂N(H)CH₂CH₂P(C₆H₅)₂

PNMeP; (C₆H₅)₂PCH₂CH₂N(CH₃)CH₂CH₂P(C₆H₅)₂

MePNMePMe; (CH₃)₂PCH₂CH₂N(CH₃)CH₂CH₂P(CH₃)₂

PNOMeP; (C₆H₅)₂PCH₂CH₂N(CH₂CH₂OCH₃)CH₂CH₂P(C₆H₅)₂

POP; (C₆H₅)₂PCH₂CH₂OCH₂CH₂P(C₆H₅)₂

PSP; (C₆H₅)₂PCH₂CH₂OCH₂CH₂P(C₆H₅)₂

Bidentate ligands L²:

O,O-cat; catecholate⁽²⁻⁾

O,O-car; carbonate⁽²⁻⁾

N,N-pda; 1,2-phenylenediaminate⁽²⁻⁾

S,S-bdt; 1,2-benzenedithiolate⁽²⁻⁾

O,O-eg; ethyleneglycolate⁽²⁻⁾

N,N-en; ethylenediaminate⁽²⁻⁾

S,S-edt; ethylenedithiolate⁽²⁻⁾

O,N-ap; 1,2-aminophenolate⁽²⁻⁾

S,N-atp; 1,2-aminothiophenolate⁽²⁻⁾

O,S-tsal; thiosalicylate⁽²⁻⁾

O,N-ae; 1,2-aminoethanolate⁽²⁻⁾

Chemistry at macroscopic (ca) level by using ⁹⁹Tc and Re

Example 1 Synthesis of mer,cis-[⁹⁹Tc(NPh)Cl₂(PNEP)][Cl]

To a solution of [Tc(NPh)Cl₃(PPh₃)₂] (45 mg) in dichloromethane/methanol(5 mL/1 mL) a 1.1 equivalent of solid PN(H)P was added under stirring.The brown mixture, in 2 minutes, turned brown-green. After 3 h thesolution was taken to dryness with a flow of nitrogen. The oily residuewas treated with diethyl ether and the resulting brown-green solidfiltered. The crude solid was washed on the filter with acetone (2 mL).The light green solid was dried under nitrogen (yield 22 mg, 60%). Theproduct is soluble in chlorinated solvents and acetonitrile, slightlysoluble in alcohols, insoluble in diethyl ether.

³¹P-NMR (300 MHz, CDCl₃, ppm): 31.6 (bs).

¹H-NMR (300 MHz, CDCl₃, ppm): 9.70 (bs, 1H; N—H), 8.06 (m, 4H, PPh₂),7.51 (m, 13H, PPh₂+NPhγ,α), 7.02 (m, 6H, PPh₂), 6.86 (t, 2H, NPhp), 3.97(bt, 2H, CH₂), 3.34 (bm, 6H, CH₂).

Example 2 Synthesis of mer,cis-[Re(NPh)Cl₂(PNHP)][Cl]

To a suspension of [Re(NPh)Cl₃(PPh₃)₂] (104 mg, 0.11 mmol) in CH₂Cl₂, anexcess of PNHP.HCl (75 mg, 0.16 mmol) dissolved in CH₂Cl₂ with 50 μl ofNEt₃ (0.38 mmol) was added dropwise. The reaction mixture was refluxedfor 2 hours and then stirred overnight at room temperature. Theresulting solution was olive-green. The solvent was removed and thegreen solid washed with diethyl ether, water and dried under vacuum. The³¹P-NMR spectrum of such solid in CDCl₃ showed two peaks at 4.3 an 8.4ppm, which suggest the presence of two new products. A pure product wasobtained by crystallization from a CH₂Cl₂/n-hexane mixture. Grey-bluecrystals were obtained (final yield 30-40%) and the compound wasidentified as [Re(NPh)Cl₂(PN(H)P)]Cl. The product is stable in air andsoluble in CH₂Cl₂, CHCl₃, quite soluble in EtOH and MeOH, insoluble inH₂O, hexane and Et₂O.

³¹P-NMR (300 MHz, CDCl₃, ppm): 8.48 (s).

¹H-NMR (300 MHz, CDCl₃, ppm): 8.07(m, 4H, PPh₂), 7.71 (d, 2H,), 7.50 (m,11H, PPh₂+NPhγ), 7.03 (m, 6H, PPh₂), 6.87 (t, 2H, NPhβ), 4.01 (bt, 2H,CH₂), 3.39 (bm, 2H, CH₂), 3.22 (bm, 4H, CH₂), 9.5 (b, 1H, NH).

Example 3 Synthesis of mer,cis-[Re(NPh)(OEt)Cl(PNHP)][Cl]

To a suspension of [Re(NPh)Cl₃(PPh₃)₂] (52 mg, 0.057 mmol) in EtOH, anexcess of PNHP.HCl (43 mg, 0.089 mmol) dissolved in EtOH with 25 μl ofNEt₃ (0.19 mmol) was added dropwise. The reaction mixture was refluxedfor 4 hours: the resulting solution was green-yellowish. The volume wasreduced under a nitrogen stream and Et₂O was added: after few hourslarge bright blue crystals of [Re(NPh)(OEt)Cl(PNHP)]Cl had been formed.

³¹P-NMR (300 MHz, CDCl₃, ppm): −0.20 (s).

¹H-NMR (300 MHz, CDCl₃, ppm): 9.5 (b, 1H, NH), 8.00 (m, 4H, PPh₂), 7.53(m, 13H, PPh₂+NPh), 7.07 (m, 6H, PPh₂), 6.87 (t, 2H, NPhp), 3.99 (bt,2H, CH₂), 3.41 (m, 2H, CH₂), 3.18 (m, 2H, CH₂), 2.98 (m, 2H, CH₂), 2.64(q, 2H; O—CH₂—CH₃), -0.13 (t, 3H; O—CH₂—CH₃).

Example 4 Synthesis of fac,cis-[Re(NPh)Cl₂(PNMeP)][Cl]

To a suspension of [Re(NPh)Cl₃(PPh₃)₂] in CH₂Cl₂ a slight excess ofPNMeP (86 mg, 0.19 mmol) was added. The reaction mixture was refluxedfor 24 h. The volume of the resulting green-yellowish solution wasreduced and Et₂O was added. A TLC analysis of the precipitate showed amixture of different products, so separation by means of acromatographic column was performed (silica gel, CHCl₃/EtOH 3/2). Twoyellow and a green fraction were collected. The green product wasidentified as [Re(NPh)Cl₂(PNMeP)]Cl.

³¹P{H}-NMR (300 MHz, CDCl₃, ppm): 19.9 (s).

¹H-NMR (300 MHz, CDCl₃, ppm): 4.08, 3.45 and 2.95 (m, 8H CH₂), 2.61 (s,3H, CH₃).

Example 5 Synthesis of mer,trans-[Re(NPh)Cl₂(PNMeP)][Cl]

[Re(NPh)Cl₃(PPh₃)₂] (100 mg, 0.11 mmol) and PNMePHCl (86 mg, 0.17 mmol)were mixed in acetonitrile. By refluxing for 15 minutes the reactionmixture became light green. Additional reflux deposited a green solidwithin a few hours. After 4 h the mixture was allowed to reach roomtemperature and filtered. The green powder was washed with Et₂O andresulted soluble in CH₂Cl₂, quite soluble in CHCl₃ and almost insolublein Et₂O and benzene. NMR analysis evidenced the presence of two isomersin solution (yield 70%), then characterised as cis-[Re(NPh)Cl₂(PNMeP)]ClHCl. ¹H-NMR (300 MHz, CD₂Cl₂, ppm): 6.59 (d); 6.74 (t); 7.45 (t) (5H,NPh); 7.10 (m); 7.24 (m); 7.37 (m); 7.55 (m); 7.70 (m); 7.85 (m); 7.96(m) (20H, PPh₂); 3.74(m); 3.35 (m); 3.08 (m) (8H, CH₂); 2.99 (d); 2.69(d) CH₃.³¹P {H} NMR (300 MHz,CD₂Cl₂,ppm): −25.1 (s); −26.9 (s). Thepale-green powder (cis-[Re(NPh)Cl₂(PNMeP)]Cl HCl) was dissolved inCH₂Cl₂ in the presence of an excess of NEt₃. NMR analysis evidenced thequantitative conversion into the mer,trans-[Re(NPh)Cl₂(PNMeP)]Clcomplex.

³¹P-NMR (300 MHz, CDCl₃,ppm): 19.9 (s)

¹H-NMR (CDCl₃, ppm)=7.6-7.1 (20H; PPh₂) 4.08, 3.45 and 2.95 (m, 8H;methylene protons); 2.61 (s, 3H; CH₃).

Example 6 Synthesis of fac-[⁹⁹Tc(NPh)(O,O-cat)(PNHP)][ClO₄]

To a solution of [⁹⁹Tc(NPh)Cl₂(PNHP)][Cl], prepared according to Example1, (26 mg, 0.04 mmol) in methanol (5 mL), catechol (5.2 mg, 0.047 mmol)and triethylamine (6.7 μL, 0.047 mmol) were added. The colour of themixture turned immediately deep purple. The solution was stirred for 4 hand concentrated to 2 mL by a nitrogen flow. A drop of a saturatedNaClO₄ solution in MeOH was added to the solution. After 1 day,dark-grey crystals were formed; they were collected on a filter andwashed with a 1×2 mL of MeOH.

³¹P-NMR (300 MHz, CDCl₃, ppm): 46.5 (bs).

¹H-NMR (300 MHz, CDCl₃, ppm): 7.77 (t, 4H), 7.49 (t, 4H,), 7.40 (t, 4H),7.22 (m, 6H) 7.02 (m, 7H), 6.87 (m, 2H), 6,71 (m, 2H), 3.5-2.7 (8H).

Example 7 Synthesis of fac-[Re(NPh)(O,O-cat)(PNHP)][Cl]

To a bluish solution of [Re(NPh)Cl₂(PNHP)]Cl, prepared according toExample 2, in CH₂Cl₂ (40 mg, 0.05 mmol) catechol (H₂cat) (5 mg, 0.045mmol) and 20 μl of NEt₃ were added at room temperature. The reactionmixture immediately turned red. The solution was stirred overnight atroom temperature. After 15 h the solvent was removed, the residue wastreated with diethyl ether and the resulting red-brown solid filteredand washed several times with n-hexane, diethyl ether and water.[Re(NPh)(O,O-cat)(PNHP)]Cl was obtained with a final yield of 53%.¹H-NMR (300 MHz, CDCl₃): 7.85-7.07 (m, 25H, NPhT and PPh₂), 6.91-6.73(m, 4H, cath). ³¹P-NMR: 19.5 (s). A stoichiometric amount of NBu₄BF₄ wasadded to a CH₂Cl₂ solution of [Re(NPh)(O,O-cat)(PNHP)]Cl and by additionof n-hexane a red-orange product precipitated. Accoprding to theelemental analysis, this powder was characterized as thetetrafluoroborate salt of [Re(NPh)(O,O-cat)(PNHP)]

³¹P-NMR: 19.5 (s).

El. Anal.: ReN₂C₄₀H₃₈O₂P₂BF₄, MW 913.7

Calcd. C 52.6, N 3.2, H 5.1

Found: C 52.9, N 3.2, H 5.1.

Example 8 Synthesis of fac-[Re(NPh)(O,O-eg)(PNHP)][Cl]

To a bluish solution of [Re(NPh)Cl₂(PNHP)]Cl of Example 2 in CH₂Cl₂ (40mg, 0.05 mmol) 30 μl of ethylene glycol and 20 μl of NEt₃ were added atroom temperature. The reaction mixture was refluxed for 1 h and thenstirred at room temperature for 24 h. The solution became grey. Afterremoval of the solvent with a gentle nitrogen stream, the oily residuewas treated with diethyl ether and the resulting grey solid filtered.The crude solid was purified washing, on the filter, with n-hexane andwater. Grey crystals of [Re(NPh)(O,O-eg)(PNHP)]Cl, suitable also forX-ray diffractometric analysis, were obtained by crystallization from aCH₂Cl₂/n-hexane solution. ³¹P {H} NMR (300 MHz, CD₂Cl₂) 17.2. ¹H-NMR(300 MHz, CDCl₃): 7.74-6.91 (m, 25H, NPh and PPh₂), 4.98 (b, 1H, NH),4.78 (d, 2H, CH₂), 4.60 (d, 2H, CH₂), 3.2-2.9 (m, 8H, PCH₂CH₂N).

Example 9 Synthesis of fac-[Re(NPh)(O,O-eg)(PNMeP)][Cl]

To a solution of fac,cis-[Re(NPh)Cl₂(PNMeP)]Cl of Example 4 in CH₃CN (50mg, 0.06 mmol) 30 μl of ethylene glycol and 20 μl of NEt₃ were added.The reaction mixture was refluxed for 24 h. The volume of the dark greensolution was reduced and the oily residue was dissolved in CH₂Cl₂, Theexcess of ethylene glycol was removed by water extraction. The organicphase was dried and a green powder, characterised asfac-[Re(NPh)(O,O-eg)(PNMeP)]Cl was recovered (yield 43%).

¹H-NMR (300 MHz, CDCl₃, 5 ppm): 6.98-7.48 (m, 25H, NPh and PPh₂);4.91-4.76 (m, 4H CH); 3.58 (m), 3.27 (m) e 2.27 (m) (8H, —CH₂CH₂—PNP;2.34 (s,3H,N—CH₃).

³¹P(CDCl₃): =24.3 (s).

Example 10 Synthesis of fac-[Re(NPh)(O,N-ap)(PNMeP)][Cl]

To a suspension of [Re(NPh)Cl₃(PPh₃)₂] in CH₂Cl₂ (90 mg, 0.1 mmol) PNMeP(53 mg, 0.11 mmol) was added at room temperature. After 5 minutes 1,2aminophenol (17 mg, 0.16 mmol) and 20 μl of NEt₃ were added and thereaction mixture was refluxed. After 10 minutes the solution turned fromgreen to dark brown. After refluxing for 30 min the solution was stirredovernight at room temperature. The solvent was removed and the residuetreated with diethyl ether. The red brown solid was filtered andre-dissolved in CH₂Cl₂. Elution from a silica column with CHCl₃/MeOH85/15 separated a yellow by-product and the redfac-[Re(NPh)(O,N-ap)(PNMeP)]Cl.

³¹P(CDCl₃): =17.8 (d), 10.1 (d).

Chemistry at microscopic (nca) level by employing ^(99m)Tc)

Example 11 Synthesis of the Intermediate [^(99m)Tc(NPh)Cl₂ L¹)]⁺Y⁻.

0.1 mL of saline physiological solution containing [^(99m)TcO₄]⁻ (50Mbq) were added to a vial containing 10 mg of PAH, 3 mg of PPh₃ 0.1 mLof 1 M HCl and 1.5 mL of MeOH. The resulting solution was heated at 65°C. for 30 min. After this, 0.2 mL of an alcoholic solution containing 3mg of an appropriate L¹ hetero-diphosphine ligand (PNHP or PNMeP orPNOMeP) was added. The resulting solution was maintained at 65° C. forfurther 30 min.

TLC analysis of the intermediate [^(99m)Tc(NPh)Cl₂L¹)]⁺ species showed amixture of different products. This behaviour is in agreement with theevidences produced at macroscopic level, where some intermediate specieswere characterised.

Example 12 Synthesis of [^(99m)Tc(NPh)L¹L²]⁺Z⁻.

This three-step preparation involves the preliminary formation of amixture of intermediate complexes of general formula [^(99m)Tc(NPh)Y₂L¹](Y=halides, water, hydroxyl, alkoxy) followed by its conversion into thefinal asymmetrical compound by reactions with the bidentate ligand L².In detail, 0.250 mL of phosphate buffer 1 M, pH 7.4, followed by 0.2 mLof methanol solution containing 5 mg of an appropriate bidentate ligandL² were added to the vial containing the intermediate compound, obtainedas reported above. The mixture was heated at 65° C. for 1 h. Theradiochemical yields evaluated by TLC chromatography are reported inTable 1. TABLE 1 Complexes TLC, Rf Yield % [^(99m)Tc(NPh)(PNHP)(O,N-ap)]0.42^(b) 62 [^(99m)Tc(NPh)(PNMeP)(O,N-ap)] 0.24^(a); 0.45^(b); 0.14^(c)97 [^(99m)Tc(NPh)(PNMeP)(S,N-atp)] 0.28^(a); 0.28^(c) 85[^(99m)Tc(NPh)(PNMeP)(O,O-cat)] 0.85^(a); 0.85^(c); 0.5^(d) 91[^(99m)Tc(NPh)(PNMeP)(S,O-tsal)] 0.78^(a); 0.75^(c), 0.35^(d) 90[^(99m)Tc(NPh)(PNMeP)(S,S-bdt)] 0.74^(a); 1^(b,c,d) 98TLC SiO₂:^(a)EtOH/CHCl₃/C₆H₆ (1/2/1.5);^(b)CHCl₃/MeOH (2% NH₄OH 20%) 85/15;^(c)CHCl₃/MeOH (2% NH₄OH 20%) 90/10;^(d)CHCl₃/MeOH (2% NH₄OH 20%) 95/5

Rf values shown by the compounds of Table 1 are in full accord with theones shown by the corresponding compounds obtained at macroscopic level,using ⁹⁹Tc, thus confirming that for the compounds of the invention thetransfer from macro to micro level is possible.

The metal-imido hetero-diphosphine complexes of the present inventionproved useful in the radiopharmaceutical field, either inradiodiagnostic imaging, when ^(99m)Tc is the employed radioactivemetal, or in radiotherapy, when ¹⁸⁶Re and ¹⁸⁸Re are the radioactivemetals.

Accordingly, the invention encompasses also the use of these complexesin the diagnostic and/or therapeutic radiopharmaceutical field and thepharmaceutical compositions comprising said compounds in admixture withpharmaceutically acceptable carriers and/or excipients.

1. A radioactive transition metal-imido hetero-diphosphine complexcompound of formula (I):[(Me═N—R)L¹L²]⁺Z⁻  (I), wherein: Me is a radioactive transition metalselected from the group consisting of ^(99m)Tc, ¹⁸⁶Re, ¹⁸⁸Re; R is aC₁-C₁₅ linear or branched alkyl or alkenyl residue, optionallyinterrupted by —O—, —S—, —N(R′)—, where R′═H or C₁-C₆ alkyl, and/oroptionally substituted with halogen, hydroxy, C₁-C₅ alkoxy, carboxy,ester, thiol, primary or secondary amino or amido, groups, or R isphenyl or an aryl residue, being R optionally substituted with abiologically active substance, wherein said biologically activesubstance is selected among sugars, amino acids, fatty acids, vitamins,hormones, peptides, catecholamines, said catecholamines being optionallyconjugated, via peptidic bond, to the other above mentioned biologicallyactive substances; L¹ is a tridentate hetero-diphosphine ligand offormula (II):

wherein: R¹, R², R³ and R⁴, which may be the same or different, have thesame meanings as R; X is oxygen, sulphur, NR⁵, wherein R⁵ is hydrogen orR; n is an integer ranging from 1 to 5; L² is a bidentate ligand, whichcomprises a combination of two donor atoms, selected from the groupconsisting of oxygen, sulphur and nitrogen, said atoms being preferablynegatively charged and being separated by a spacer of 2 to 4 members,said spacer being an aliphatic chain or part of an aromatic ring, L²being optionally conjugated to a biologically active substance as abovedefined; Z⁻ is a mononegative counter-ion selected from the groupconsisting of Cl⁻, Br⁻, OH⁻, ClO₄ ⁻, EtO⁻, tetrafluoroborate.
 2. Aradioactive transition metal-imido hetero-diphosphine complex accordingto claim 1, wherein the radioactive transition metal is ^(99m)Tc.
 3. Aradioactive transition metal-imido hetero-diphosphine complex accordingto claim 1, wherein R is selected from the group consisting of methyl,ethyl, propyl, isopropyl, butyl, isobutyl, octyl, decyl, dodecyl,propenyl, butenyl, pentenyl, phenyl, benzyl, tolyl, 4-methoxy-benzyl,4-ethoxy-benzyl, salicyl.
 4. A radioactive transition metal-imidohetero-diphosphine complex according to claim 3, wherein R issubstituted with a biologically active substance, said substance being acatecholamine selected from the group consisting of dopamine, L-DOPA,3-hydroxythyramine, optionally conjugated, via peptide bond, to anotherbiologically active substance selected from the group consisting ofsugars amino acids, fatty acids, vitamins, hormones, peptides, andcatecholamines.
 5. A complex according to claim 4, wherein dopamine isconjugated to vitamin H.
 6. A radioactive transition metal-imidohetero-diphosphine complex according to claim 1, wherein L¹ is selectedfrom the group consisting of: (C₆H₅)₂PCH₂CH₂N(H)CH₂CH₂P(C₆H₅)₂;(C₆H₅)₂PCH₂CH₂N(CH₃)CH₂CH₂P(C₆H₅)₂;(C₆H₅)₂PCH₂CH₂N(CH₂CH₂OCH₃)CH₂CH₂P(C₆H₅)₂;(CH₃)₂PCH₂CH₂N(CH₃)CH₂CH₂P(CH₃)₂; (C₆H₅)₂PCH₂CH₂SCH₂CH₂P(C₆H₅)₂;(C₆H₅)₂PCH₂CH₂OCH₂CH₂P(C₆H₅)₂;
 7. A radioactive transition metal-imidohetero-diphosphine complex according to claim 1, wherein L² comprises acombination of two electron-donor atoms selected from the groupconsisting of [O⁻,O⁻], [N⁻,O⁻], [S⁻,O⁻], [N⁻,N⁻], [N⁻,S⁻] and [S⁻,S⁻],said atoms being separated by a 2 to 4 membered spacer, wherein saidspacer is an aliphatic chain or part of an aromatic ring.
 8. A complexaccording to claim 7, wherein L² is selected from the group consistingof catecholate⁽²⁻⁾; carbonate⁽²⁻⁾; 1,2-phenylenediaminate⁽²⁻⁾;1,2-benzenedithiolate⁽²⁻⁾; ethyleneglycolate⁽²⁻⁾; ethylenediaminate⁽²⁻⁾;ethylenedithiolate⁽²⁻⁾; 1,2-aminophenolate⁽²⁻⁾;1,2-aminothiophenolate⁽²⁻⁾; thiosalicilate⁽²⁻⁾; 1,2-aminoethanolate⁽²⁻⁾.9. A complex according to claim 7, wherein L² is conjugated to acatecholamine selected from the group consisting of dopamine, L-DOPA,3-hydroxythyramine, optionally conjugated to another biologically activesubstance selected from the group consisting of sugars, amino acidsfatty acids, vitamins, hormones, peptides, and catecholamines.
 10. Acomplex according to claim 9, wherein dopamine is conjugated to vitaminH.
 11. A radioactive transition metal-imido hetero-diphosphine complexaccording to claim 1, wherein Z⁻ is Cl⁻, ClO₄ ⁻, EtO⁻,tetrafluoroborate.
 12. A process for the preparation of the radioactivecompounds of formula (I) comprising the following steps: reacting anoxide of a transition metal MeO₄ ⁻ with an excess of tertiarymonophosphine, in a hydro-alcoholic solution acidified with hydrochloricacid and in the presence of 1-substituted-2-acetyl hydrazine, to give animido complex of formula (IV):[(Me═N—R)Y₃(PPh₃)₂]  (IV), wherein: Me is ^(99m)TcO₄ ⁻, ¹⁸⁶ReO₄ ⁻,¹⁸⁸ReO₄ ⁻; R is as defined in claim 1; Y is Cl, Br, OH, reacting saidcompound of formula (IV) with a tridentate heterodiphosphine ligand L¹,in organic solvents selected from alcohols, chlorinated solvents,acetonitrile or a mixture thereof, optionally in the presence of anorganic base, at a temperature ranging from room temperature to thereflux temperature of the solvent, to give the intermediate compound offormula (III):[(Me═N—R)Y₂L¹)]⁺Y⁻  (III), wherein: Me, R, Y, are as defined above, andL¹ is a tridentate hetero-diphosphine ligand of formula (II):

wherein: R¹, R², R³ and R⁴, which may be the same or different, have thesame meanings as R; X is oxygen, sulphur, NR⁵, wherein R⁵ is hydrogen orR; n is an integer ranging from 1 to 5, and reacting said intermediatecompound of formula (III) with a bidentate ligand L² in alcoholicsolution, after adjusting pH at 7.4 with phosphate buffer.
 13. A processaccording to claim 12, wherein said 1-substituted-2-acetyl hydrazine is1-phenyl-2-acetyl hydrazine.
 14. A process according to claim 12,wherein the oxide of the transition metal is ^(99m)TcO.
 15. Anintermediate compound of formula (III):[(Me═N—R)Y₂L¹)]⁺Y⁻  (III), wherein Me is ^(99m)TcO₄ ⁻, ¹⁸⁶ReO₄ ⁻,¹⁸⁸ReO₄ ⁻; R is as defined in claim 1; Y is Cl, Br, OH, and L¹ is atridentate hetero-diphosphine ligand of formula (II):

wherein: R¹, R², R³ and R⁴, which may be the same or different, have thesame meanings as R; X is oxygen, sulphur, NR⁵, wherein R⁵ is hydrogen orR; and n is an integer ranging from 1 to
 5. 16. A radioactive transitionmetal-imido hetero-diphosphine complex of claim 1 for use inradiodiagnostic imaging.
 17. A radioactive transition metal-imidohetero-diphosphine complex of claim 1 for use in radiotherapy.
 18. Apharmaceutical composition comprising a radioactive transitionmetal-imido hetero-diphosphine complex of claim 1 in admixture withpharmaceutically acceptable carriers and/or excipients.